pH and Kinetic Isotope Effects on Sarcosine Oxidation by N-Methyltryptophan Oxidase

Abstract

N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/K_O2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k_cat/K_sarc pH profile is bell-shaped, with pK_a values of 8.8 and about 10; the latter value matches the pK_a value of the substrate nitrogen. The k_cat pH profile exhibits a single pK_a value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k_cat/K_sarc value. With N-methyl-²H₃-glycine as the substrate, there is a pH-independent kinetic isotope effect on k_cat, k_cat/K_sarc, and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme−substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.